To investigate the contribution and mechanism of eosinophil granulocytes and NK cells-mediated cytotoxicity to the pathogenesis of ITP. Mononuclear cells and platelets were prepared from the bone marrow of 16 ITP patients and 10 healthy controls. Separately, eosinophil granulocytes and NK cells were selected with magnetic microbeads. As the target cells, the autologous platelets were cultured with eosinophil granulocytes and NK cells respectively for 6 h and then stained with annexin V. Ratio of platelets expressing annexin V was determined by flow cytometry. The fraction of NK cells expressing perforin, granzyme B, FasL and TNF were determined by flow cytometry. Human eosinophil cationic protein (ECP) level in the bone marrow was determined by ECP ELISA kit. The annexin V positive platelet ratio of the ITP group was significantly higher than that of the control group; the expression rates of granzyme B, perforin, FasL on NK cells of the ITP group were significantly higher than those of the control group. Expression of human ECP in bone marrow of immune thrombocytopenia patients was higher than that of healthy controls. NK cells and eosinophil granulocytes are activated in ITP and might be involved in the pathogenesis of ITP.